PB0361 - Coxsackievirus B3 infection of CD34+ cells impairs megakaryocyte and platelet production through activation of Toll-like receptors 7 and 8

Background: Increasing evidence indicates that hematopoietic progenitor cells (CD34+ cells), megakaryocytes (MKs), and platelets (PLT) express toll-like receptors (TLRs) allowing the contribution of these cells to the immune response and inflammation. However, whether TLRs’ activation on CD34+ modulates megakaryo/thrombopoiesis during viral infections is still unclear.

Aims: We evaluated whether the single-strand RNA Coxsackievirus B3 (CVB3) infection modulates human MK development and PLT production through TLR7/8 pathway.

Methods: CD34+ cells derived from human umbilical cord blood were exposed to CVB3 or UV-irradiated CVB3 and then stimulated with thrombopoietin in the presence or absence of TLR7/8 antagonists. TLRs expression, proliferation rate, CD34* differentiation, MK maturation, and PLT biogenesis were determined by flow cytometry and mRNA of Fli-1, RUNX-1, NF-E2, and IFN-beta by qPCR.

Results: CD34+ cells express both TLR7 and TLR8 (Fig. 1A-B). Exposure of cells to CVB3 resulted in productive infection as determined by the presence of viral infectious particles in culture supernatants after 24h and 11 dpi (Fig. 1C). Total cell number, differentiation towards MKs (CD41+cells), MK maturation (CD42b and CD42a+cells), and platelet biogenesis, were markedly reduced (n=6, p< 0.05) in infected cultures (Fig.1 D-H). The mRNA expression of transcription factors involved in these processes including Fli-1, RUNX-1, and NF-E2, was also downregulated in infected cells (n=5, p< 0.05) (Fig 1I-K). The reduction in MK growth was not due to alteration of cell proliferation but was associated with an increase in cell apoptosis and the RNA levels of IFN-beta(Fig 2 A-C). Decreased cell number, MK maturity, PLT production, and apoptosis were significantly reversed by TLR7/8 antagonists (Fig. 2 D-G) and partially mimicked by Imiquimod, an agonist of TLR7/TLR8 (n=4, p< 0.05).

Conclusion(s): These data suggest a new role for TLR7/8 in megakaryo/thrombopoiesis during viral infections that might contribute to a better understanding of the molecular bases underlying thrombocytopenia during infectious processes.